Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome

Abstract

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.

Figure 1

Schematic representation of EZ∷TN5 transposome preparation. A) pYV02, the transposon carrying plasmid: mosaic end left (MEL), erythromycin resistance gene (ermF), kanamycin resistance gene (Km), E. coli conditional origin of replication (R6Kori), mosaic end right (MER), E. coli origin of replication (ColE1 ori), ampicillin resistance gene (amp). B) Digestion of pYV02 with PvuII/PshAI which cuts near mosaic ends yield the EZ∷TN5 transposon. C) Incubation of transposon with EZ∷TN5 transposase (•) yields the transposome (which is then used for electroporation).

Figure 2

Transposition of transposome in BF638R. Southern hybridization: DNA from transposon mutant was digested with BglII and transferred to a nylon membrane. The ermF gene present on chromosomal DNA was probed with the biotin Chromogenic kit. Lane 1. M Biotinylated 2-Log DNA Ladder, Lanes 2-7 are EZY mutants.

Figure 3

Identification of mutated gene in BF638R. A) Schematic representation of SRP-PCR. The first round PCR was done with SRP1 and EzTnSeqR1 with template DNA from the mutant. The second round PCR was done with SRP2 and EzTnSeqR2 primers with 1μl template from the first round PCR. The product of second round PCR was column purified and sequenced with the EzTnSeqR3 primer.

Figure 4

Transposon mutants of BF638R. There are 4,535 genes in the BF638R genome. Twenty random transposon mutants were identified by SRP-PCR and named EZY-4 thru 21. The locus tag of the gene in which the transposon was inserted is shown.